The percentage of hospital-acquired infections caused by staphylococci has increased over the past ten years. Staphylococci are now the most common cause of primary nosocomial bacteremias. A major factor in this increase has been the emergence of nosocomial staphylococci resistant to multiple antimicrobial agents. Coincident with this increase in resistance has been the description of new antimicrobial resistance genes and new systems for the dissemination is the aim of the current proposal. First, we propose to continue studies on the genetic basis of conjugative plasmid transfer. Conjugative plasmids are newly described in staphylococci and the mechanism for their self-transfer is unknown. We have defined a single contiguous region of DNA both necessary and sufficient for transfer. Cloned fragments of this tra region and transposon insertions that abolish transfer will now be used in trans- complementation studies to define genetic organization. Surface- expressed tra proteins will be identified by polyclonal antibody, their role in mating pair formation or surface exclusion assessed, and their genes located using insertion mutants and cloned tra fragments. Finally, regions of DNA and gene products that mediate the following activities will be identified: the origin of transfer, plasmid mobilization, and a newly-discovered protoplast lethality function. The second aim of this proposal will be to study the role of insertion- sequence (IS)-like elements in the genesis and evolution of conjugative plasmids and in the mobility of antimicrobial resistance genes. We have found that these sequences bound antimicrobial resistance genes and the tra region. Their independent mobility, role in DNA translocation, and contribution to gene expression will be assessed by cloning, DNA/DNA hybridization and DNA sequencing. The movement of resistance genes found to be associated with these sequences in clinical isolates will help define their biologic role in the construction of the multi-resistant phenotype. Taken together, these two aspects of the proposal will help to define factors responsible for the dissemination of antimicrobial resistance genes both within and between staphylococci.